This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Two metalloproteins containing alkaline earth metal binding sites were investigated in this project: class C acid phosphatases and parvalbumins. Class C acid phosphatases (CCAPs) are outer membrane acid phosphatases that contain four essential Asp residues imbedded in the bipartite sequence motif of [IV]-[VAL]-D-[IL]-D-E-T-[VM]-L-X-[NT]-X(2)-Y in the N-terminal half of the polypeptide chain and [IV]-[LM]-X(2)-G-D-[NT]-L-X-D-F in the C-terminal half. The active site features a magnesium ion bound to three of the conserved Asp residues. Because of their localization to the bacterial outer membrane, CCAPs are potential candidates for vaccine development, and some progress has been made toward creating a vaccine against nontypeable Haemophilus influenzae using catalytically inactive mutants of the H. influenzae CCAP. We are studying the structure, function, and immunogenicity of CCAPs from pathogenic bacteria including H. influenzae, F. tularensis, and B. anthracis in order to understand the role of these enzymes in virulence and to provide a platform for the design of inactive mutants with enhanced immunogenicity for use in recombinant vaccines. The second aspect of this project focuses on parvalbumins, which are small (Mr=12,000), vertebrate-specific EF-hand proteins. The parvalbumin family includes alpha and beta sublineages, distinguished by isoelectric point and lineage-specific sequence differences. Despite the general similarity of their metal-ion binding sites and overall fold, parvalbumins exhibit broad variations in divalent ion affinity. We are exploring the physical and structural basis for these differences, using specific parvalbumin isoforms.